Building on a Solid Foundation: Adding Relevance and Reproducibility to Neurological Modeling Using Human Pluripotent Stem Cells

Abstract

The brain is our most complex and least understood organ. Animal models have long been the most versatile tools available to dissect brain form and function; however, the human brain is highly distinct from that of standard model organisms. In addition to existing models, access to human brain cells and tissues is essential to reach new frontiers in our understanding of the human brain and how to intervene therapeutically in the face of disease or injury. In this review, we discuss current and developing culture models of human neural tissue, outlining advantages over animal models and key challenges that remain to be overcome. Our principal focus is on advances in engineering neural cells and tissue constructs from human pluripotent stem cells (PSCs), though primary human cell and slice culture are also discussed. By highlighting studies that combine animal models and human neural cell culture techniques, we endeavor to demonstrate that clever use of these orthogonal model systems produces more reproducible, physiological, and clinically relevant data than either approach alone. We provide examples across a range of topics in neuroscience research including brain development, injury, and cancer, neurodegenerative diseases, and psychiatric conditions. Finally, as testing of PSC-derived neurons for cell replacement therapy progresses, we touch on the advancements that are needed to make this a clinical mainstay.

Keywords: disease modeling; human brain development; human pluripotent stem cells; neural stem cells; regenerative medicine.

Figure 1

Animal and cell culture models fall on a continuum that balances providing physiological complexity with human brain relevance. Non-mammalian model organisms like worms (C. elegans) and fruit flies (D. melanogaster) allow molecular and functional investigation of simple neuronal circuits and rational dissection of critical regulatory pathways in a physiologically relevant setting. Rodent and non-human primate models provide additional physiological complexity by permitting quantitative analysis of complex behavioral traits and simultaneously enrich relevance to the human condition. Human subjects and dissected human brain tissues fall high on both measures, but their procurement is rare or ethically impossible. The emergence of human pluripotent stem cell (hPSC)-derived 2D cell and 3D tissue models offer a highly needed source of neurological modeling tools with high relevance to the human brain. Though they offer less physiological complexity than animal models this capacity is ever increasing as technology advances (defined cell co-cultures and 3D tissue engineering). Furthermore, their relevance to the human condition is greatly enhanced compared to traditional culture of immortalized or non-human primary cell lines. A combination of hPSC-based and animal model approaches offers the best opportunity to capture both physiological complexity and relevance to the human brain.

Figure 2

Advances in hPSC-based neurological modeling are increasingly improving the complexity of human neural cell types and circuits we can model. Somatic cells from multiple human patient sources (fibroblast skin cells, blood, urine) can now be used to produce induced human pluripotent stem cells (hPSCs) by over-expression of typically four key pluripotency transcription factors (OCT4, SOX2, KLF4, c-MYC). Similarly, induced neurons can be directly established by over-expression of neuronal identity genes like NGN2. It is essential to regularly perform quality control assays (detailed in the main text) to ensure maintenance of a high-quality pluripotent state in reprogrammed hPSCs. This stage also offers a critical period for genome editing (e.g., CRISPR-Cas9) in which to introduce mutations of interest or create isogenic control lines. As a field, we can reproducibly generate fetal-stage excitatory and inhibitory neurons in 2D and 3D cell cultures. These cultures permit analysis of cell-autonomous mechanisms of development and disease, especially in the presence of a disease-causing mutation. Recent efforts have increased our ability to generate additional specialized brain-resident cells (“Heterogenous brain cell types”) and to produce more mature neurons (purple = excitatory and inhibitory neurons with elaborate neurite networks; green = astrocytes; orange = oligodendrocytes; yellow = microglia). These heterogeneous cultures permit modeling of non-cell autonomous processes. Offering the most advanced level of complexity in hPSC models are 3D brain tissue engineering approaches, including organoids (rounded enclosed objects = ventricular zones; blue shading = neural stem and progenitor cells; green shading = neurons and other differentiated cell types) and bioprinted constructs, which can incorporate microfluidic channels for advanced microenvironmental control. Studies have also begun to incorporate vasculature and components of the blood-brain barrier into microfluidic and organoid models. The box to the right outlines recent and developing strategies for promoting neuronal maturation and aging in 2D and 3D models.